Immune-deficient animals to study "hormone-dependent" breast and endometrial cancer

Athymic (nu/nu) mice are T cell deficient and can accept xenografts of human tumor material. Hormone-dependent tumor growth can be demonstrated in ovariectomized athymic mice by estrogen administration. Estrogen receptor (ER) positive MCF-7 breast cancer cells implanted into the axillary mammary fat do not grow into palpable tumors unless sustained release preparations of estrogen are administered. The non-steroidal antiestrogen tamoxifen, though it exhibits estrogenic properties in the mouse, does not facilitate MCF-7 tumor growth (during short term, i.e. 8 weeks of therapy) and can prevent estradiol-stimulated growth. In contrast, ER negative MDA-MB-231 cells grow with or without estrogen administration and tamoxifen does not control tumor growth. These statements reflect current dogma concerning the value of athymic mice to confirm the hormone dependent growth of cancer cells in vivo. Our aim has been to define the limits of this dogma and to investigate the growth relationship of hormone-dependent and independent cells with their host environment. The potential endocrine or paracine effect of ER negative tumors on the growth of ER positive tumors was evaluated by transplantation on opposite sides of athymic mice or by the inoculation of different ratios of ER positive/negative cells (MCF-7:MDA-MB-231 9:1, 99:1, 999:1). MCF-7 cells could not be encouraged to grow by a rapidly growing MDA-MB-231 tumor on the opposite side of the animal. Similarly ER negative tumors grew out of the mixed tumor inoculates suggesting that ER positive tumors could not be encouraged to grow preferentially by the paracrine influences of ER negative cells. However, estrogen facilitates the growth of an ER positive tumor following inoculation of mixed cell populations. Antiestrogen treatment can blunt estrogen-stimulated growth but cannot control the growth of ER positive/negative containing tumors. ER positive endometrial tumors grow in response to estrogen treatment and some (EnCa101) have been shown to grow in response to tamoxifen or a combination of tamoxifen and estrogen. More unusual though is our recent observation that an ER negative primary endometrial tumor (BR) and its metastasis (BR-MET) grow more rapidly in estrogen-treated athymic mice. This finding seems to have far-ranging consequences for our view of hormone-dependent growth. Either our view of estrogen-stimulated growth needs to be modified or the host is specifically altered during estrogen treatment. We have taken the position that since natural killer cells (present in athymic mice) can be lowered by estrogen this may result in an increased tumor cell survival in the heterotransplant model.(ABSTRACT TRUNCATED AT 400 WORDS)     

The channel properties of possible gramicidin dimers

Extending previous work (Sung & Jordan, 1987 a, Biophys. J. 51, 661-672; 1988, Biophys. J.54, 519-526), we describe channel properties of five possible gramicidin dimers by studying dimerization energies and axial electrical potentials. Unlike the head-to-head dimer (the predominant channel former), both tail-to-tail and head-to-tail dimers with the same beta-helical monomer structure as the head-to-head dimer only form four intermonomer hydrogen bonds and are much less stable. Were channels formed from these dimers to be observed, their electrical potential profiles suggest that they should be cation selective, probably conduct less than the head-to-head dimer, have a central cation binding site, bind cations preferentially if crystallizable, and in the case of the head-to-tail dimer, rectify. Like the antiparallel double stranded helical dimer (a possible minor conducting pathway) the parallel double stranded helical dimer has 28 interstrand hydrogen bonds, but its hydrogen bond network is quite distorted and it is much less stable. If it formed, its electrical potential profile suggests that it would be cation selective, bind anions preferentially if crystallizable, rectify, and at high enough voltages, might exhibit a conductance greater than that of the antiparallel form.     

An immunochemical assay model system for the sensitive detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit pyruvate dehydrogenase (E1).

An immunochemical enzyme immunoassay model system was developed and compared for maximum sensitivity with a radioimmunoassay method and the classic enzyme activity method for the detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit, pyruvate dehydrogenase (E1), isolated from Escherichia coli. Cross-linked large molecular weight antibody-enzyme conjugate systems are compared with heterobifunctional singular antibody conjugates substituted with high levels of horseradish peroxidase. Both polyclonal and monoclonal antibodies generated to the Escherichia coli PDHc and E1 antigens were used to develop a double-antibody sandwich microtiter plate enzyme-linked immunosorbent assay. It is demonstrated that a double sandwich immunochemical assay system can be quantitative for PDHc, can detect PDHc in crude cell lysates and has levels of sensitivity of 2.0.10(-16) mol for the detection of PDHc. This assay model system provides specific antibody selection criteria and coupling methods needed to select specific antisera that cross-react with human PDHc. This rapid and sensitive immunochemical assay method clearly demonstrates that sensitive mass assay systems can be developed for the detection of PDHc. Different from Western blot, this methodology could be used to generate mass assays which could be applied to the rapid detection of mammalian antigens (employing the corresponding antibodies) implicated in a number of pyruvate dehydrogenase deficiencies associated with human disorders.     

Mobilizable carbon reserves in young peach trees as evidenced by trunk girdling experiments

The seasonal patterns in concentrations of both soluble (NSC-S)and insoluble (NSC-I) non-structural carbohydrates, in 3-year-oldpeach trees (Prunus persica L. Batsch) grown in sand cultureare described. The ability of trees to mobilize their carbohydratereserves in response to scion-trunk girdling, which preventsphotosynthate transport toward the roots, was tested at fourphenological stages. Girdling induces a NSC-I depletion in rootsand rootstock-trunk bark and a NSC-I accumulation in leavesand shoots. On the contrary, the NSC-S concentrations of theorgans located both above and below girdling were not significantlyaffected by the treatment. Consequently, when phloem transportbreaks down, trees, whatever their growing stage, mobilize carbohydratereserves below the girdle to maintain the soluble sugar contentsat the same level as in control trees. Key words: Girdling, non-structural carbohydrates, Prunus persica L., carbon reserves, seasonal patterns     

Complete replication in vitro of tobacco mosaic virus RNA by a template-dependent, membrane-bound RNA polymerase.

A crude membrane-bound RNA polymerase, obtained by differential centrifugation of extracts of tomato leaves infected with tobacco mosaic tobamovirus (tomato strain L) TMV-L), was purified by sucrose density gradient centrifugation. Removal of the endogenous RNA template with micrococcal nuclease rendered the polymerase template dependent and template specific. The polymerase was primer independent and able to initiate RNA synthesis on templates containing the 3'-terminal sequences of the TMV-L positive or negative strands. TMV-vulgare RNA was a less efficient template, while RNAs of cucumber mosaic cucumovirus and red clover necrotic mosaic dianthovirus, or 5'-terminal sequences of TMV-L positive or negative strands, did not act as templates for the polymerase. A main product of the reaction with TMV-L genomic RNA as a template, carried out in the presence of [alpha-32P]UTP, was genomic-length single-stranded RNA. This was shown to be the positive strand and uniformly labelled along its length, demonstrating complete replication of TMV-L RNA. Genomic-length double-stranded RNA, labelled in both strands, and small amounts of RNAs corresponding to the single- and double-stranded forms of the coat protein subgenomic mRNA were also formed. Antibodies to N-terminal and C-terminal portions of the 126-kDa protein detected the 126-kDa protein and the 183-kDa readthrough protein in purified RNA polymerase preparations, whereas antibodies to the readthrough portion of the 183-kDa protein detected only the 183-kDa protein. All three antibodies inhibited the template-dependent RNA polymerase, but none of them had any effect on the template-bound enzyme.     

Transmission of feline immunodeficiency virus in domestic cats via artificial insemination.

The objective of this study was to determine whether semen from male domestic cats infected with feline immunodeficiency virus (FIV) can transmit virus to females. Twelve inseminations were performed by an intrauterine laparoscopic technique with fresh or cryopreserved electroejaculates from asymptomatic males chronically infected with the NCSU1 strain of FIV. Of six inseminations performed with fresh semen, three resulted in infection of queens, as indicated by seroconversion, expression of FIV gag provirus in peripheral blood leukocytes, and reduced peripheral CD4+/CD8+ T-lymphocyte ratios. None of the six inseminates with thawed cryopreserved semen resulted in infection. Two infected queens and one uninfected queen became pregnant. Virus was not evident in the seven offspring. We conclude that FIV can be transmitted horizontally by artificial insemination with fresh semen.     

K252a Modulates the Expression of Nerve Growth Factor-Dependent Capsaicin Sensitivity and Substance P Levels in Cultured Adult Rat Dorsal Root Ganglion Neurones

Abstract: K252a, an inhibitor of trk phosphorylation and nerve growth factor signal transduction in PC12 cells, blocked nerve growth factor-induced responses in cultured adult rat dorsal root ganglion sensory neurones. The nerve growth factor-dependent appearance of capsaicin sensitivity and accumulation of the neuropeptide substance P were inhibited when dorsal root ganglion neurones were grown in the presence of low concentrations (100 n M ) of K252a. At higher concentrations (3 µ M ), however, K252a stimulated the development of capsaicin sensitivity and the accumulation of substance P even in the absence of nerve growth factor. By using a wide dose range, therefore, we showed that K252a could either inhibit or mimic nerve growth factor's actions on sensory neurones. These results may explain the apparent paradox in the literature that some groups show a blocking effect of K252a on nerve growth factor-dependent survival of dorsal root ganglion sensory neurones, whereas others report that K252a can substitute for nerve growth factor or other trophic factors and promote neuronal survival.     

Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation.

DNA-calcium phosphate co-precipitates arise spontaneously in supersaturated solutions. Highly effective precipitates for transfection purposes, however, can be generated only in a very narrow range of physico-chemical conditions that control the initiation and growth of precipitate complexes. The concentrations of calcium and phosphate are the main factors influencing characteristics of the precipitate complex, but other parameters, such as temperature, DNA concentration and reaction time are important as well. An example for this is the finding that almost all of the soluble DNA in the reaction mix can be bound into an insoluble complex with calcium phosphate in <1 min. Extending the reaction time to 20 min results in aggregation and/or growth of particles and reduces the level of expression. With improved protocols we gained better reproducibility and higher efficiencies both for transient and for stable transfections. Up to 60% of cells stained positive for beta-gal and transient production of secreted proteins was improved 5- to 10-fold over results seen with transfections using standard procedures. Similar improvements in efficiency (number of recombinant cell colonies) were observed with stable transfections, using co-transfected marker plasmids for selection. Transient expression levels 2 days after DNA transfer and titers obtained from stable cell lines, emerging weeks later, showed strong correlation.     

Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5 end of the cDNA was amplified by a 5-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.